臺灣博碩士論文加值系統

本研究將Bacillus halodurans的xyn45基因片段基因重組在pET29a質體上轉殖於大腸桿菌E. coli BL21(DE3)。重組大腸桿菌培養於PM培養基進行高密度醱酵並持續進行饋料，並加入IPTG誘導使其表現內切型木聚醣水解酶。在0.10 mM/OD600 IPTG誘導18小時後，發現胞外蛋白質比活性平均為11.6 ± 0.4 U/mg。高密度醱酵時，為了達到細胞高密度而持續饋料，造成醱酵培養基中的鹽類濃度過高，明顯會抑制酵素活性。因此，透過離心濃縮管以及超濾法透析後去除金屬離子，可克服鹽類的抑制作用。與原始酵素液相比，去除離子之後酵素液的比活性平均可提升36%。
在酵素應用方面，本研究使用之木質纖維素的原料為蔗渣。以鹼液進行前處理之後，使用三種方式萃取出半纖維素，萃取率由高到低分別為酒精沉澱法、超濾法以及兩步驟法。將萃取得到的半纖維素與Xyn45 基因重組酵素在不同pH值之下進行水解反應，得到木寡糖(XOS)。酒精沉澱法在pH 7.5時，木寡糖產率為25.0%；超濾法及兩步驟法在pH 7時，則分別達到27.5%及30.4%的木寡糖產率。

Extracellular endo-1,4-β-xylanase encoded by the xyn45 gene from Bacillus halodurans was over-expressed in recombinant Escherichia coli BL21 (DE3) using plasmid pET29a as the vector. The recombinant E. coli was cultivated to high-cell-density by fed-batch fermentation in PM medium and IPTG was used to induce the synthesis of this recombinant endoxylanase. As 0.10 mM of IPTG per OD (600 nm) of cells was used for the induction, the extracellular endoxylanase with a specific activity of 11.6 U/mg in the final culture medium was obtained after induced 18 hour. In the fed-batch fermentation to achieve a high cell density, salts could be fed too much and result in significantly inhibition of the endoxylanase activity by high salt concentrations in the fermentation medium. Desalting of the enzyme preparation by dialysis to removal metal ions could increase about 36% in the specific activity.
For the application of recombinant enzyme for producing xyligoligoccharides (XOS), xylan was prepared from the alkaline solution of sugarcane bagasse by three kinds of method. The xylan extraction yield from high to low was found by precipitation with ethanol, ultrafiltration, and then two-step method. The resultant hemicellulose (xylan) was then incubated with enzyme preparation in different pH values for XOS production. The XOS mixture contained a total of 25.0% (w/w) of xylobiose and xylotriose as the xylan was prepared by ethanol precipitation in pH 7.5. The XOS yields were 27.5% and 30.4%, respectively, when ultrafiltration and two-step method were used in pH 7.

致謝 I
中文摘要 II
Abstract III
目錄 IV
圖目錄 VIII
表目錄 X
第一章 緒論 1
1.1 前言 1
1.2 研究目的 2
第二章 文獻回顧 3
2.1 大腸桿菌 3
2.2 木質纖維素 (Lignocellulose) 4
2.2.1 纖維素 (Cellulose) 4
2.2.2 半纖維素 (hemicellolose) 6
2.2.3 木質素(Lignin) 6
2.3 鹼處理 (Alkaline pretreatment) 7
2.4 蔗渣(Sugarcane bagasse, SCB) 8
2.5 木聚醣水解酶 (Xylanase) 9
2.6 木寡糖 (Xylooligosaccharide, XOS) 12
第三章 實驗材料與方法 13
3.1 實驗藥品 13
3.1.1 重組菌的培養及誘導 13
3.1.2 高壓破菌 14
3.1.3 蛋白質定量及活性測定 14
3.1.4 SDS-PAGE 14
3.1.5 鹼處理法萃取木聚醣 15
3.1.6 組成分析 15
3.2 實驗儀器與材料 16
3.2.1 儀器 16
3.2.2 材料 18
3.3 菌種來源及培養基 19
3.3.1 菌種來源 19
3.3.2 培養基 19
3.4 重組菌培養及誘導 19
3.4.1 塗盤培養 19
3.4.2 前培養 19
3.4.3 放大50 ml 20
3.4.4 高密度培養 (High-Cell-Density Cultivation, HCDC) 20
3.5 酵素樣品處理 23
3.6 SDS-PAGE 24
3.7 活性測定方法(Bailey et al., 1992) 26
3.8 蛋白質定量 29
3.9 透析方法 30
3.9.1 超濾法 (Ultrafiltration, UF) 30
3.9.2 離心濃縮管 (Centrifugal Concentrators, CC) 31
3.10 組成分析 32
3.10.1 萃取率測定 32
3.10.2 碳水化合物分析 33
3.10.3 酸不可溶木質素分析 34
3.11 實驗架構 35
3.12 木聚醣之製備 36
3.12.1 酒精沉澱 36
3.12.2 超濾法 (Ultrafiltration, UF) 36
3.12.3 兩步驟法（先超濾法再酒精沉澱） 37
3.13 木寡糖(Xylooligosaccharide) 37
第四章 實驗結果與討論 38
4.1 搖瓶培養 38
4.2 高密度醱酵 40
4.2.1 生長曲線 40
4.2.2 活性分析及蛋白質濃度測定 41
4.2.3 高密度醱酵SDS-PAGE 42
4.2.4 透析結果 43
4.3 蔗渣組成 46
4.3.1 蔗渣萃取所得之半纖維素組成 48
4.4 木聚醣之萃取率 49
4.5 酵素水解反應 51
4.5.1 酒精沉澱法萃取的基質 51
4.5.2 超濾法萃取的基質 51
4.5.3 兩步驟法萃取的基質 52
第五章 結論與建議 65
5.1 結論 65
5.2 建議 66
第六章 參考文獻 67
附錄Ａ、Xyn45序列資料(李, 2012) 72
附錄B、HPLC檢量線 74
附錄C、高密度醱酵重複實驗 76

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